Amino acid acylase

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

L-Amino acid acylases hydrolyze the amide bonds of A-acetyl-L-amino acids (Equation (9)). 

Figure 12.3-5. Coupled enzymatic reaction of a dehydro amino acid acylase with a amino acid dehydrogenase (from1 21).

Effect of metal ions on the catalytic activity of amino acid acylase isolated from alpha-amylase complex... 

Amino acid acylase is the bio-catalyst that is used in selective hydrolysis of acetyl L-amino acids to L-amino acids from acetyl DL-amino acid racemic mixtures. In this study tlie effect of metal ions like Co, Ni, Mn and Ca on the activity of amino acid acylase which was isolated from alpha-amylase complex (Sanzyme) was investigated. It was observed that in presence of Co ions the activity of enzyme increased by 48% where as when Ca Ni" and Mn ions were added the activity decreased by 5%, 20% and 37% respectively. 

Keywords Alpha-amylase complex. Amino acid acylase. Acetyl methionine. Metal ions and Ninhydrin. 

Amino acid acylases have wide distribution in nature. They are found in animal tissues like kidney [1], molds, bacteria, yeast, and its activity is traced in plants including mushrooms. They serve as effective tools in resolution of L-amino acids from DL-amino acid racemic mixtures. L-amino acids are naturally occurring, physiologically active amino acids which are the building blocks of all the proteins. 

In an attempt to find a suitable source of amino acid acylase, Sanzyme, essentially an alpha-amylase complex extracted from a strain of Aspergillus oryzae was explored for the presence of this enzyme. Aspergillus oryzae is known to contain amylase, however, there is no report available for the presence of this enzyme in Sanzyme strain. It was found out that the crude Sanzyme samples contain 7.8 units of amino acylase activity [2], where one unit is defined as 1 micro mole of acetyl methionine hydrolysed/hr/mg of enzyme. It was also found out that the purified form of amino acylase from Sanzyme contain 28.4 units of activity [3] which is high when compared to any other amino acylase reported in literature. 

From Fig-1 the activity of purified amino acid acylase from Sanzyme is calculated as 28.4 units where 1 unit is defined as 1 micro mole of acetyl methionine hydrolysed/hr/mg of enzyme. This activity of native enzyme is taken as 100% and it is compared with the activity of enzyme in presence of metal ions. From Fig-1 and Table-1 it can be observed that the activity of amino acylase in presence of Co" ions is increased by 48% when compared to native enzyme. In presence of Ni" and Ca ions the activity of enzyme is reduced by 20%... 

Fig. 1. Comparison of activity of amino acid acylase in native form in presence of Co ions on selective hydrolysis of acetyl DL - methionine.

From the above results it can be observed that Co ions increase the activity of amino acid acylase to a high extent. This is possible by binding of Co ions at the active site of enzyme due to which the stability of enzyme-substrate complex increases which leads to... 

The fact that Co ions increase the activity of amino acid acylase in resolution of L-methionine from acetyl DL-methionine, shall be applied advantageously in the practical process of enzymatic resolution of DL-methionine. Usually in practical resolution, as cost of enzyme is high, it is desirable to employ a higher substrate concentration and less amount of enzyme which leads to production of L-methionine vith less cost. From the above results it can be concluded that by using Co ions we can reduce the amount of enzyme to a greater extent in enzymatic resolution of L-methionine by using amino acid acylase isolated from Sanzyme. 

L-Amino acid acylase from Aspergillus melleus (Amano acylase 30000) was used to hydrolyze racemic 6-acetoxybuspirone to (5)-6-hydroxybuspirone 2 in 96% enantiomeric excess (EE) after 46% conversion. The remaining (l )-6-acetoxy-buspirone with 84% EE was converted to (l )-6-hydroxybuspirone 2 by acid hydrolysis [28]. The EE of both enantiomers could be improved to more than 99% by crystallization as a metastable polymorph. Direct hydroxylation of buspirone to (5 )-6-hydroxbuspirone by Streptomyces antibioticus ATCC 14980 has also been described [28]. 

In 1935, Krebs published a technique for Isomer analysis that employs D-amino acid oxidase, an enzyme which selectively deaminates D-isomers. This method allowed measurement of D-amino acids via the resultant keto acids that were formed or by recovery of intact L-amino acids (28). Other enzymatic methods based on L-amino acid decarboxylases (25,43), L-amino acid acylases and amidases (44) also have been used. Other biologically-based techniques employing selective utilization of L-amino acids by microorganisms appeared as early as 1949 (29,30), but due to their complexity, have not been used widely. 

Aspergillus oryzae Cellulose nitrate Amino acid acylase L-Tryptophan... 

Amino acid acylase Porcine kidney, Aspergillus melleus... 

L-Amino acid acylase from kidneys DL-Acylamino acid — L-Amino acid + D-Acyl-amino acid Production of essential amino aids for human and animal nutrition. 

The chemical synthesis of amino acids leads to a racemic mixture. Only the L-form is usable for medicines and foodstuffs. Immobilized enzymes have been used for the industrial separation of enantiomers of various amino acids, e.g., amino acid acylase immobilized onto DEAE-Sephadex. Processing efficiency in the fermentation of amino acids has been greatly facilitated through the use of immobilized amino acid acylases such as L-glutamate aminoacylase. 

Figure 6.28 Process for the resolution of DL-methionine with amino acid acylase...

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